Hematopoietic stem cells (HSCs) are derived during embryogenesis and proliferate and differentiate to form the blood lineages. Recent experiments in our lab have defined the genesis of adult HSCs in the aorta-gonad-mesonephros (AGM) of the developing zebrafish. This provides a visual system to study pathways that regulate hematopoietic stem cell development. Here we plan to make use of this system to examine pathways and treatments that perturb HSC formation and homeostasis. From the earliest studies characterizing HSCs, their unique sensitivity to DNA damaging agents such as ionizing radiation and alkylating agents has been appreciated. These agents act through the ATM-p53 DNA damage response pathway, which is critical for sensing double stranded DNA breaks and initiating a program of apoptosis. In mouse, ATM has an essential function in the self-renewal of hematopoietic stem cells during normal development. Loss of p53 has been implicated in HSC self-renewal in mouse bone marrow. ATM and p53 regulate pathways which appear to control survival and death decisions for HSCs during development. I hypothesize that loss of ATM and p53 confer distinct defects in HSC development. Using the zebrafish I propose to study the effects of loss of p53 or ATM on HSC during normal development by in situ analysis of HSC markers. We will determine if p53 or ATM loss compromise the ability of HSCs to respond to DNA damaging treatments. We propose to create a zebrafish line with ATM deficiency to determine the requirements for ATM in HSC self-renewal. These studies will clarify the role that p53 and ATM play in normal HSC development. Using the p53 mutant zebrafish I propose a genetic suppressor screen to identify novel genes involved in protecting HSCs from DNA damaging agents. I plan to clone 1-2 genes identified in the screen and characterize them by testing their ability to interact with other pathways which regulate HSC development. These genes identified in this screen will offer valuable insights into the biology of stem cells, and will be useful as HSCs are used in transplantation for diseases such as sickle cell anemia and aplastic anemia.